TRPC3 ion channel subunit immunolocalization in the cochlea

Canonical transient receptor potential (TRPC) subunits assemble as tetramers to form ion channels with high calcium (Ca²⁺) permeability. Here, we investigated the possibility that TRPC3 ion channels are broadly expressed in the adult guinea pig and mouse cochleae. Using immunofluorescence, pronounced labeling occurred in the spiral ganglion (SG) neurons, inner hair cells (IHC), outer hair cells (OHC) and epithelial cells lining scala media. TRPC3 expression was homogeneous in the SG throughout the cochlea. In contrast, there was marked spatial variation in the immunolabeling in the cochlear hair cells with respect to location. This likely relates to the tonotopy of these cells. TRPC3 immunolabeling was more pronounced in the IHC than OHC. Both basal region IHC and OHC had higher TRPC3 expression levels than the corresponding cells from the apical region of the cochlea. These data suggest that TRPC3 ion channels contribute to Ca²⁺ homeostasis associated with the hair cells, with higher ion fluxes in more basal regions of the cochlea, and may also be a significant pathway for Ca²⁺ entry associated with auditory neurotransmission via the SG neurons. TRPC3 expression was also identified within the spiral limbus region, inner and outer sulcus, but without evidence for spatial variation in expression level. Expression in these gap junction-coupled epithelial cells lining scala media is indicative of a contribution of TRPC3 channels to cochlear electrochemical homeostasis.     

Maturation of neurotransmission in the developing rat cochlea: immunohistochemical evidence from differential expression of synaptophysin and synaptobrevin 2

Synaptophysin and synaptobrevin 2 associate closely with packaging and storage of synaptic vesicles and transmitter release, and both play important roles in the development of rat cochlea. We examined the differential expression of synaptophysin and synaptobrevin 2 in the developing Sprague-Dawley rat cochlea, and investigated the relationship between their expression and auditory development. The expression of synaptophysin and synaptobrevin 2 was not observed in Kolliker’s and Corti’s organ at postnatal 1 day (P1) and P5, and the top turn of the cochlea at P10. Expression was detected in the outer spiral bundle (OSB), the inner spiral bundle (ISB), and the medial wall of the Deiters’ cell of the cochlea at P14, and P28, and in the middle or the basal turn of Corti’s organ at P10. Synaptobrevin 2 was expressed in the top of the inner hair cells (IHCs) in Corti’s organ of both P14 and P28 rats. All spiral ganglion neurons (SGNs) were stained at all ages examined. The localization of synaptophysin and synaptobrevin 2 in the cochlea was closely associated with the distribution of nerve fibers and neural activity (the docking and release of synaptic vesicles). Synaptophysin and synaptobrevin 2 were expressed in a dynamic manner during the development of rat cochlea. Their expression differences during the development were in favor of the configuration course constructed between nerve endings and target cells. It also played a key role in the formation of the correct coding of auditory information during auditory system development.     

Upregulation of HSP60 expression in the postnatal rat cochlea and rats with drug-induced hearing loss

Heat shock protein 60 (HSP60) is a highly conserved chaperone molecule that plays important roles in mediating some physiological and pathological functions. However, researchers have not yet determined whether HSP60 is expressed in the mammalian cochlea. This study constitutes the first investigation of the expression of HSP60 in the postnatal rat cochlea. We also examined the expression of HSP60 in rats with drug-induced hearing loss. Auditory thresholds were assessed by monitoring the auditory brainstem response (ABR) prior to and after drug injection. Expression levels of the HSP60 gene (Hsp60) and HSP60 protein in the rat cochlea were detected by quantitative real-time polymerase chain reaction and Western blotting, respectively. The distribution of HSP60 in the rat cochlea was further examined by immunofluorescence staining. We have demonstrated that HSP60 was expressed in the postnatal rat cochlea in an age-dependent and cell-specific manner. In addition, after drug exposure, the average hearing threshold of rats in the experimental group was significantly higher than that in the control group, with increased HSP60 expression level in response to kanamycin and furosemide treatments. HSP60 expression was observed in the supporting cells (SCs) within the organ of Corti in both the uninjured and the injured cochlea, but it was undetectable in the mechanosensory hair cells (HCs) and spiral ganglion neurons. Therefore, our research suggests that HSP60 may play an important role in auditory function.     

Reinnervation of hair cells by auditory neurons after selective removal of spiral ganglion neurons

Hearing loss can be caused by primary degeneration of spiral ganglion neurons or by secondary degeneration of these neurons after hair cell loss. The replacement of auditory neurons would be an important step in any attempt to restore auditory function in patients with damaged inner ear neurons or hair cells. Application of beta-bungarotoxin, a toxin derived from snake venom, to an explant of the cochlea eradicates spiral ganglion neurons while sparing the other cochlear cell types. The toxin was found to bind to the neurons and to cause apoptotic cell death without affecting hair cells or other inner ear cell types as indicated by TUNEL staining, and, thus, the toxin provides a highly specific means of deafferentation of hair cells. We therefore used the denervated organ of Corti for the study of neuronal regeneration and synaptogenesis with hair cells and found that spiral ganglion neurons obtained from the cochlea of an untreated newborn mouse reinnervated hair cells in the toxin-treated organ of Corti and expressed synaptic vesicle markers at points of contact with hair cells. These findings suggest that it may be possible to replace degenerated neurons by grafting new cells into the organ of Corti.     

Expression of Growth Factors and Their Receptors in the Postnatal Rat Cochlea

RT-PCR was used to assay for growth factors and receptors from seven different protein families in cochlea tissues of the juvenile rat. There was a broad representation of the growth factor families in all the cochlea tissues examined, though the organ of Corti and stria vascularis expressed a greater variety than the spiral ganglion. This broad expression suggests that a variety of known growth factors play significant roles in the development, maintenance, and repair of the inner ear. The results of this survey serve as a basis for the design of future in vitro experiments that will address the ability of growth factors to protect hair cells from damage and to evoke a repair-regeneration response by injured hair cells.     

Expression and function of pannexins in the inner ear and hearing

Pannexin (Panx) is a gene family encoding gap junction proteins in vertebrates. So far, three isoforms (Panx1, 2 and 3) have been identified. All of three Panx isoforms express in the cochlea with distinct expression patterns. Panx1 expresses in the cochlea extensively, including the spiral limbus, the organ of Corti, and the cochlear lateral wall, whereas Panx2 and Panx3 restrict to the basal cells of the stria vascularis in the lateral wall and the cochlear bony structure, respectively. However, there is no pannexin expression in auditory sensory hair cells. Recent studies demonstrated that like connexin gap junction gene, Panx1 deficiency causes hearing loss. Panx1 channels dominate ATP release in the cochlea. Deletion of Panx1 abolishes ATP release in the cochlea and reduces endocochlear potential (EP), auditory receptor current/potential, and active cochlear amplification. Panx1 deficiency in the cochlea also activates caspase-3 cell apoptotic pathway leading to cell degeneration. These new findings suggest that pannexins have a critical role in the cochlea in regard to hearing. However, detailed information about pannexin function in the cochlea and Panx mutation induced hearing loss still remain largely undetermined. Further studies are required.     

Developmentally regulated expression of ectonucleotidases NTPDase5 and NTPDase6 and UDP-responsive P2Y receptors in the rat cochlea

Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) regulate complex extracellular P2 receptor signalling pathways in mammalian tissues by hydrolysing extracellular nucleotides to the respective nucleosides. All enzymes from this family (NTPDase1-8) are expressed in the adult rat cochlea. This study reports the changes in expression of NTPDase5 and NTPDase6 in the developing rat cochlea. These two intracellular members of the E-NTPDase family can be released in a soluble form and show preference for nucleoside 5′-diphosphates, such as UDP and GDP. Here, we demonstrate differential spatial and temporal patterns for NTPDase5 and NTPDase6 expression during cochlear development, which are indicative of both cytosolic and extracellular action via pyrimidines. NTPDase5 is noted during the early postnatal period in developing sensory hair cells and supporting Deiters’ cells of the organ of Corti, and primary auditory neurons located in the spiral ganglion. In contrast, NTPDase6 is confined to the embryonic and early postnatal hair cell bundles. NTPDase6 immunolocalisation in the developing cochlea underpins its putative role in hair cell bundle development, probably via cytosolic action, whilst NTPDase5 may have a broader extracellular role in the development of sensory and neural tissues in the rat cochlea. Both NTPDase5 and NTPDase6 colocalize with UDP-preferring P2Y₄, P2Y₆ and P2Y₁₄ receptors during cochlear development, but this strong association was lost in the adult cochlea. Spatiotemporal topographic expression of NTPDase5 and NTPDase6 and P2Y receptors in adult and developing cochlear tissues provide strong support for the role of pyrimidinergic signalling in cochlear development.     

Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea

The adult mammalian cochlea receives dual afferent innervation: the inner sensory hair cells are innervated exclusively by type I spiral ganglion neurons (SGN), whereas the sensory outer hair cells are innervated by type II SGN. We have characterized the spatiotemporal reorganization of the dual afferent innervation pattern as it is established in the developing mouse cochlea. This reorganization occurs during the first postnatal week just before the onset of hearing. Our data reveal three distinct phases in the development of the afferent innervation of the organ of Corti: (1) neurite growth and extension of both classes of afferents to all hair cells (E18-P0); (2) neurite refinement, with formation of the outer spiral bundles innervating outer hair cells (P0-P3); (3) neurite retraction and synaptic pruning to eliminate type I SGN innervation of outer hair cells, while retaining their innervation of inner hair cells (P3-P6). The characterization of this developmental innervation pattern was made possible by the finding that tetramethylrhodamine-conjugated dextran (TMRD) specifically labeled type I SGN. Peripherin and choline-acetyltransferase immunofluorescence confirmed the type II and efferent innervation patterns, respectively, and verified the specificity of the type I SGN neurites labeled by TMRD. These findings define the precise spatiotemporal neurite reorganization of the two afferent nerve fiber populations in the cochlea, which is crucial for auditory neurotransmission. This reorganization also establishes the cochlea as a model system for studying CNS synapse development, plasticity and elimination.     

Expression of EFR3A in the Mouse Cochlea during Degeneration of Spiral Ganglion following Hair Cell Loss

Retrograde degeneration of spiral ganglion cells in the cochlea following hair cell loss is similar to dying back in pathology. The EFR3A gene has recently been discovered to be involved in the pathogenesis of dying back. The relationship of EFR3A and spiral ganglion degeneration, however, was rarely investigated. In this study, we destroyed the hair cells of the mouse cochlea by co-administration of kanamycin and furosemide and then investigated the EFR3A expression during the induced spiral ganglion cell degeneration. Our results revealed that co-administration of kanamycin and furosemide quickly induced hair cell loss in the C57BL/6J mice and then resulted in progressive degeneration of the spiral ganglion beginning at day 5 following drug administration. The number of the spiral ganglion cells began to decrease at day 15. The expression of EFR3A increased remarkably in the spiral ganglion at day 5 and then decreased to near normal level within the next 10 days. Our study suggested that the change of EFR3A expression in the spiral ganglion was coincident with the time of the spiral ganglion degeneration, which implied that high expression of EFR3A may be important to prompt initiation of spiral ganglion degeneration following hair cell loss.     

Expression and distribution of creatine transporter and creatine kinase (brain isoform) in developing and mature rat cochlear tissues

Physiological processes in the cochlea associated with sound transduction and maintenance of the unique electrochemical environment are metabolically demanding. Creatine maintains ATP homeostasis by providing high-energy phosphates for ATP regeneration which is catalyzed by creatine kinase (CK). Cellular uptake of creatine requires a specific high affinity sodium- and chloride-dependent creatine transporter (CRT). This study postulates that this CRT is developmentally regulated in the rat cochlea. CRT expression was measured by quantitative real-time RT-PCR and immunohistochemistry in the postnatal (P0–P14) and adult (P22–P56) rat cochlea. The maximum CRT expression was reached at the onset of hearing (P12), and this level was maintained through to adulthood. CRT immunoreactivity was strongest in the sensory inner hair cells, supporting cells and the spiral ganglion neurons. Cochlear distribution of the CK brain isoform (CKB) was also assessed by immunohistochemistry and compared with the distribution of CRT in the developing and adult cochlea. CKB was immunolocalized in the organ of Corti supporting cells, and the lateral wall tissues involved in K⁺ cycling, including stria vascularis and spiral ligament fibrocytes. Similar to CRT, CKB reached peak expression after the onset of hearing. Differential spatial and temporal expression of CRT and CK in cochlear tissues during development may reflect differential requirements for creatine–phosphocreatine buffering to replenish ATP consumed during energy-dependent metabolic processes, especially around the period when the cochlea becomes responsive to airborne sound.     

Fibroblast growth factor-2 immunoreactivity is present in the central and peripheral auditory pathways of adult rats

Recent findings have pointed out the role of neurotrophic factors in the survival and maintenance of neurons of the auditory system. Basic fibroblast growth factor (bFGF, FGF-2) is a potent neurotrophic molecule whose actions can be seen in the central and peripheral nervous systems. In the present study, FGF-2 immunoreactivity was analyzed in the auditory pathways of the adult rat, employing a well-characterized polyclonal antibody against FGF-2. In the cochlea, FGF-2 immunoreactivity was observed in the inner and outer hair cells of the organ of Corti, spiral ganglion neurons, spiral limbus, and stria vascularis. Stereological methods employing optical fractionator revealed the presence of 84.5, 15, and 0.5% of spiral ganglion neurons possessing FGF-2 immunoreactivity of strong, moderate, and weak intensity, respectively. In the central auditory pathways, FGF-2 immunoreactivity was found in the cytoplasm of the neurons of the cochlear nuclei, trapezoid body nuclei, medial geniculate nucleus, and inferior colliculus. The two-color immunoperoxidase method showed FGF-2 immunoreactivity in the nuclei of astrocytes throughout the central auditory pathway. Computer-assisted microdensitometric image analysis revealed higher levels of specific mean gray values of FGF-2 immunoreactivity in the trapezoid body and ventral cochlear nucleus and also in the spiral ganglion and inner hair cells. Sections incubated with FGF-2 antibody preabsorbed with human recombinant FGF-2 showed no immunoreaction in the majority of the studied regions, exhibiting only a slight immunoreactive product in the hair cells of the organ of Corti. Furthermore, no changes in immunoreactivity were observed in sections incubated with FGF-2 antiserum preincubated with human recombinant acidic FGF (FGF-1). The findings suggest that FGF-2 may exert paracrine and autocrine actions on neurons of the central and peripheral auditory systems and may be of importance in the mechanism of hearing diseases.     

NTPDase1 and NTPDase2 immunolocalization in mouse cochlea: implications for regulation of p2 receptor signaling.

Cellular, molecular, and physiological studies have demonstrated an important signaling role for ATP and related nucleotides acting via P2 receptors in the cochlea of the inner ear. Signal modulation is facilitated by ectonucleotidases, a heterologous family of surface-located enzymes involved in extracellular nucleotide hydrolysis. Our previous studies have implicated CD39/NTPDase1 and CD39L1/NTPDase2, members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, as major ATP-hydrolyzing enzymes in the tissues lining the cochlear endolymphatic and perilymphatic compartments. NTPDase1 hydrolyzes both nucleoside triphosphates and diphosphates. In contrast, NTPDase2 is a preferential nucleoside triphosphatase. This study characterizes expression of these E-NTPDases in the mouse cochlea by immunohistochemistry. NTPDase1 can be immunolocalized to the cochlear vasculature and neural tissues (primary auditory neurons in the spiral ganglion). In contrast, NTPDase2 immunolabeling was principally localized to synaptic regions of the sensory inner and outer hair cells, stereocilia and cuticular plates of the outer hair cells, supporting cells of the organ of Corti (Deiters' cells and inner border cells), efferent nerve fibers located in the intraganglionic spiral bundle, and in the outer sulcus and root region of the spiral ligament. This differential expression of NTPDase1 and 2 in the cochlea suggests spatial regulation of P2 receptor signaling, potentially involving different nucleotide species and hydrolysis kinetics.     

Developmental regulation of TRPC3 ion channel expression in the mouse cochlea

Canonical transient receptor potential type 3 (TRPC3) ion channels assemble from TRPC3 subunits and exhibit multiple activation mechanisms. TRPC3 has been proposed to contribute to Ca²⁺ entry supporting Ca²⁺ homeostasis in cochlear hair cells and to be activated by G protein-coupled receptor (GPCR) signaling in spiral ganglion neurons. The present study was designed to determine the spatiotemporal profile of TRPC3 expression during mouse cochlear ontogeny. TRPC3 immunofluorescence of cryosectioned cochleae was performed using E16–adult tissue. We found that prior to birth, TRPC3 expression was strongest in epithelial cells that form the cochlear partition. In the early postnatal period, to the onset of hearing (~P12), immunofluorescence was strongest in the hair cells, with increased expression in stria vascularis and Reissner’s membrane. Afferent neurite labeling in inner spiral plexus and outer spiral bundles developed transiently in the perinatal period, corresponding to the critical period of synaptic consolidation, while signal in the spiral ganglion soma increased from the perinatal period through to adulthood. Compared with the late embryonic/early postnatal levels, hair cell expression was relatively weaker from the third postnatal week, whereas spiral ganglion soma labeling was stronger. In the adult, TRPC3 expression was primarily in the soma of spiral ganglion neurons, the hair cells, and the inner and outer sulcus regions. This spatiotemporal profile of TRPC3 expression was consistent with this ion channel contributing to development of sensory, neural and epithelial cochlear tissues, as well as hair cell Ca²⁺ homeostasis and regulation of auditory neurotransmission via GPCR signaling.